The present invention relates generally to the fields of infection and disease. More particularly, it concerns the identification of a new spirochete carried by the hard tick, Amblyomma americanum, found by the present inventor to be associated with a Lyme disease-like illness in the southeastern and south-central United States. Most particularly, the invention provides compositions, methods, and kits for the identification of the new spirochete for diagnostic purposes.
A paradox about Lyme disease is the report of this tick-borne infection from areas in which transmission of the etiologic agent, B. burgdorferi, has not been documented (Sigal et al., 1991; Barbour et al., 1993). This phenomenon has been reported from Georgia and Missouri, but may be common in other parts of the southeastern and south-central United States (Centers for Disease Control and Prevention, 1989; 1991). The Lyme disease-like illness is a localized, expanding circular skin rash, sometimes succeeded by persistent, debilitating systemic symptoms (Masters, 1993; Donnell, 1992). Many of the patients with this illness have had negative serologic assays for antibodies to B. burgdorferi, a finding that has fueled a controversy about so-called xe2x80x9cseronegative Lyme diseasexe2x80x9d (Sigal et al., 1991; Barbour et al., 1993). Although Ixodes scapularis ticks, the usual vector of the Lyme disease agent, has been identified in some of these geographic areas, the more commonly reported exposure for these patients has been to another hard tick, A. americanum, known as the xe2x80x9cLone Star tickxe2x80x9d (Centers for Disease Control and Prevention 1989; 1991; Masters, 1993; Donnell, 1992). One conclusion from these observations is that the disease is caused by something other than B. burgdorferi and that the vector of the putative agent is A. americanum (Maupin et al., 1992).
The incompetence of A. americanum as a vector of B. burgdorferi has been documented (Piesman et al., 1988; Mather et al., 1990; Mukolwe et al., 1992; Ryder et al., 1992). Nevertheless, there have been descriptions in these ticks of spirochetes that cross-react with antibodies to the Lyme disease agents (Maupin et al., 1992; Schulze et al., 1984). Until the discovery of B. burgdorferi and related Borrelia species in Ixodes spp. ticks a decade ago, Borrelia spp. had almost exclusively been found in soft or argasid ticks (Barbour et al., 1986).
Reports from several locations in the southeastern and south-central regions of the United States indicate that this Lyme disease-like illness, which is apparently ameliorated by antibiotics, is associated with bites by the Lone Star tick (Centers for Disease Control and Prevention, 1989; 1991; Masters, 1993; Donnell, 1992). A. americanum is a common person-biting tick in these areas (Cooney et al., 1974; Koch et al., 1980; Hair et al., 1986; Bloemer et al., 1990). Its usual hosts are white-tailed deer, medium-sized mammals, and ground-feeding birds; rodents are only rarely infested by A. americanum. The tick""s distribution extends from west-central Texas to Florida and north to Rhode Island (Cooney et al., 1974; Koch et al., 1980; Hair et al., 1986; Bloemer et al., 1990).
Numerous references in the literature relate to aspects of diagnosing and treating Lyme disease. For example: i) U.S. Pat. No. 5,279,938 relates to a nucleotide sequence of a recombinant clone containing a specific segment of Borrelia burgdorferi (Bb) DNA, the causative agent of Lyme disease; ii) an abstract by Barthhold (WPI Acc. No.: 92-041321/05) relates to OSPA polypeptides immuno-reactive with antibodies generated by the spirochete Borrelia burgdorferi; iii) The Weisburg world patent publication relates to nucleic acid fragments that are used to detect the etiological agent of Lyme disease, Borrelia; iv) The Oliver et al. (1993) abstract relates to a study of the isolation and transmission of the Lyme disease spirochete; v) The Berland et al. (1991) abstract relates to the characterization of a 41 kDa flagellin antigen of B. burgdorferi; vi) The Mukolwe et al. (1992) article relates to attempts to transmit the B. burgdorferi (Bb) spirochete to three different ticks, one of these being the Amblyomma americanum tick. The test results report transfer of the Bb spirochete only to Ixodes scapularis ticks.
Although there is much known about Lyme disease, there are currently no means of identification of the new spirochete associated with the aforedescribed Lyme disease-like pathology and further, no means of diagnosis of infection, compositions for clinical tests, or laboratory assays for diagnosing a patient exhibiting Lyme disease-like symptoms but testing negative for Lyme disease.
The present invention provides compositions, methods, and kits for the detection of a new spirochete that is associated with a Lyme disease-like illness. The compositions are based on a Borrelia lonestari sp. nov.-specific allotype or combination of allotypes of the flagellin protein, or a Borrelia lonestari sp. nov.-specific allele or combination of alleles of the flagellin or 16s rRNA genes of the new spirochete. The allotypes and alleles provided by the present invention have been determined by nucleic acid sequencing of portions of the flagellin and rRNA genes from this new spirochete. Detection of a species-specific amino acid or nucleotide as defined herein, or a species-specific combination of amino acids or nucleotides as defined herein, in a subject sample is indicative of infection with Borrelia lonestari sp. nov.
xe2x80x9cSpecies-specific allotypexe2x80x9d or xe2x80x9cspecies-specific amino acidxe2x80x9d or xe2x80x9cspecies-specific epitopexe2x80x9d means an amino acid of B. lonestari sp. nov. that is different at a particular position of the flagellin protein amino acid sequence than the amino acid at that position of the flagellin protein of other Borrelia species, especially those species needing to be distinguished from B. lonestari sp. nov. Table 1 provides a listing of species-specific amino acids of this new spirochete in the context of the amino acid sequence of SEQ ID NO: 2.
xe2x80x9cSpecies-specific combination of allotypesxe2x80x9d or xe2x80x9cspecies-specific combination of amino acidsxe2x80x9d or xe2x80x9cspecies-specific combination of epitopesxe2x80x9d is a combination of amino acids of the flagellin protein of B. lonestari sp. nov. from Table 1 that is not represented in any of the flagellin proteins of other Borrelia species, especially those species needing to be distinguished from B. lonestari sp. nov. Table 1 also provides a listing of amino acids that may be combined with each other to form a combination that is unique to B. lonestari sp. nov. in the context of the amino acid sequence of SEQ ID NO: 2.
xe2x80x9cSpecies-specific allelexe2x80x9d or xe2x80x9cspecies-specific nucleotidexe2x80x9d means a nucleotide of B. lonestari sp. nov. that is different at a particular position of the flagellin gene sequence or 16s rRNA gene sequence from the nucleotide at that position of other flagellin gene sequences or 16s rRNA gene sequences of Borrelia species, especially the Borrelia species that need to be particularly distinguished, like B. burgdorferi. Tables 2 and 3 provide a listing of species-specific nucleotides of this new spirochete in the context of SEQ ID NO: 1 and 3.
xe2x80x9cSpecies-specific combination of allelesxe2x80x9d or xe2x80x9cspecies-specific combination of nucleotidesxe2x80x9d is a combination of nucleotides of the flagellin gene or 16s rRNA gene of B. lonestari sp. nov. from Table 2 or 3 that is not represented in any of the flagellin gene sequences or 16s rRNA gene sequences of other Borrelia species. Tables 2 and 3 provide a listing of nucleotides that may be combined with each other to form a combination that is unique to B. lonestari sp. nov. in the context of SEQ ID NO: 1 and 3.
Species-specific flagellin amino acids of B. lonestari sp. nov. are listed in Table 1 as the underlined residues in the column Bl and include Val 24, Thr 65, Ala 67, Phe 90, Ser 91, Thr 92, Gly 99, Val 103, Pro 119, Ile 126, Ser 127, Ile 136, Ala 140, Thr 144, Asp 174, and Ile 191, of SEQ ID NO:2.
Species-specific flagellin nucleotides of B. lonestari sp. nov. are listed in Table 2 as the underlined nucleotides in the column Bl and include G 70, G 96, T 141, A 193, G 199, G 228, A 231, T 269, C 270, T 271, A 273, A 300, T 308, G 315, A 376, G 380, A 406, G 418, G 423, G 505, A 510, G 546, T 572, and C 603 of SEQ ID NO:1.
Exemplary species-specific combinations of amino acids where the amino acid itself is not species-specific are found by comparing the amino acids of Table 1 and finding a combination of Bl amino acids that is not represented in any of the other species listed in the context of the flagellin amino acid sequences of these organisms. Examples include: amino acid #s 41 and 46, 46 and 108, 117 and 153, 130 and 153, 46 and 147, 152 and 169, 152 and 171, and 46 and 196 of SEQ ID NO:2, for example.
Of course, Tables 1 and 2 clearly demonstrate the differences in amino acids and nucleotides of the flagellin proteins and genes of B. lonestari sp. nov. and B. burgdorferi, the causative agent of Lyme disease in North America (Barbour and Fish, 1993) and the most relevant organism to distinguish B. lonestari sp. nov. from in a diagnostic test.
Exemplary species-specific combinations of nucleotides where the nucleotide itself is not species-specific are found by comparing the nucleotides of Table 2 and finding a combination of Bl nucleotides that is not represented in any of the other species listed in the context of the sequence of SEQ ID NO: 1. Examples include: nucleotide NT #30 and 225, 42 and 225, 177 and 297, 303 and 312, 350 and 355, 375 and 419, 432 and 435, 458 and 475, and 501 and 516 of SEQ ID NO:1, for example. With these examples, one skilled in the art would, upon further examination of Table 2, find further species-specific combinations of nucleotides in the context of SEQ ID NO: 1 for identification of B. lonestari sp. nov.
An embodiment of the present invention is a purified nucleic acid molecule comprising a nucleotide sequence of about 12 to about 709 nucleotides that encodes a B. lonestari sp. nov. flagellin peptide having at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids from Table 1, or a complement thereof. In a preferred embodiment, the nucleotide sequence has the sequence of SEQ ID NO:1, 4 or 26. An even more preferred embodiment is a purified nucleic acid molecule having a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO: 2, a partial sequence of the B. lonestari sp. nov. flagellin protein.
Further embodiments include a recombinant molecule comprising the nucleic acid molecule described above, a host cell comprising the recombinant molecule and the recombinant molecule is preferably an expression vector. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
The at least one B. lonestari sp. nov. specific amino acid may be at position 24, 65, 67, 90, 91, 92, 99, 103, 119, 126, 127, 136, 140, 174, or 191 of SEQ ID NO:2 as shown in Table 1. The at least one B. lonestari sp. nov.-specific combination of amino acids is also obtained from Table 1 as described above.
The at least one B. lonestari sp. nov.-specific amino acid or combination of amino acids can be considered an allotype of this species. Preferably, the length of the oligonucleotide is from about 12 to about 641 nucleotides; or in other embodiments, from about 12 to about 330 nucleotides; or 12 to about 300; or 12 to about 150; or 12 to about 99; and in still other embodiments, from about 15 to about 30 nucleotides. In other embodiments, the nucleotide sequence encodes amino acid(s) at and flanking position 24, 65, 67, 90, 91, 92, 99, 103, 119, 126, 127, 136, 140, 174, or 191 of SEQ ID NO:2. Preferably, the sequence encodes amino acids at and flanking positions 90-92, 103-108, 119-127, 136-144, or 171-174 of SEQ ID NO:2. In another embodiment, the sequence encodes a species-specific combination of amino acids of Table 1 having flanking amino acids from SEQ ID NO:2. The oligonucleotide may be defined further as including a detectable label. Some oligonucleotides may be defined further as comprising the sequence GGTGTTCAAGCG, SEQ ID NO:7 or GTTCAACCAGCT, SEQ ID NO:8. These sequences are unique to B. lonestari sp. nov. due to the presence of a number of nucleotides at particular positions around 310 and 358 of the flagellin gene of other Borrelia species. These species-specific oligonucleotides are useful as hybridization probes for the detection of B. lonestari sp. nov. in a diagnostic assay.
A further embodiment of the invention is a purified nucleic acid molecule comprising a nucleotide sequence represented in SEQ ID NO:1 or 3 having at least one B. lonestari sp. nov.-specific nucleotide or species-specific combination of nucleotides from Table 2 or 3, or a complement thereof. Another embodiment is a purified flagellin gene of B. lonestari sp. nov. A further embodiment of the present invention is a nucleic acid segment that comprises at least a 10-14 nucleotide long stretch that corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:1 and includes an allele as described in Table 2. In a more preferred embodiment, the nucleic acid is further defined as comprising at least about a 20 nucleotide long stretch, about 30 nucleotide long stretch, about 50 nucleotide long stretch, about 100 nucleotide long stretch, about 200 nucleotide long stretch, about 400 nucleotide long stretch, about 600 nucleotide long stretch, or a full length sequence that corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:1 and includes an allele as described in Table 2.
The present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1, 3, 4, 26 or other of the segments described herein. Nucleic acid sequences which are xe2x80x9ccomplementaryxe2x80x9d are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term xe2x80x9ccomplementary sequencesxe2x80x9d means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleqtide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:1, 3, 4 or 26 under relatively stringent conditions such as those described herein. The B. lonestari sp. nov. nucleotides set forth in Tables 2 and 3, however, are considered relatively invariant since they are species-specific or a combination of the nucleotides is species-specific.
A purified nucleic acid molecule comprising a nucleotide sequence encoding a B. lonestari sp. nov. 16s ribosomal RNA is a further embodiment of the present invention. Preferably, the nucleotide sequence has a sequence comprising SEQ ID NO:3. The nucleic acid may be defined further as a recombinant molecule.
A preferred embodiment of the present invention is a purified flagellin protein of B. lonestari sp. nov. The protein may be defined further as an amino acid sequence comprising SEQ ID NO:2. The term xe2x80x9cthe amino acid sequence of SEQ ID NO:2xe2x80x9d means that the sequence substantially corresponds to a portion of SEQ ID NO:2 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein as having the amino acids of SEQ ID NO:2 listed in Table 1, these amino acids being relatively invariant in their function as species-specific epitopes or combination of epitopes of B. lonestari sp. nov. The flagellin protein or portions thereof having species-specific epitopes or a combination of epitopes is useful in an immunoassay for the detection of B. lonestari sp. nov.
A purified peptide having an amino acid sequence comprising about 6 to about 213 amino acids of SEQ ID NO:2 that includes at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids from Table 1 is a further embodiment of the present invention. Preferably, the peptide has from about 6 to about 212 amino acids; more preferably, from about 6 to about 150 amino acids; and in other embodiments, from about 6 to about 50 amino acids. The above-described peptide preferably includes B. lonestari sp. nov. specific amino acid(s) at and flanking position 24, 65, 67, 90, 91, 92, 99, 103, 119, 126, 127, 136, 140, 174, or 191 of SEQ ID NO:2. Preferably, the peptide includes amino acid(s) at and flanking positions 90-92, 103-108, 119-127, 136-144, or 171-174 of SEQ ID NO:2. In another embodiment, the peptide includes a species-specific combination of amino acids of Table 1 having flanking amino acids from SEQ ID NO:2. In some embodiments, the peptide may include a detectable label. Preferred peptides comprise the sequence Gly Val Gln Ala, SEQ ID NO:5 or the sequence Val Gln Pro. These sequences are unique to B. lonestari sp. nov. due to the presence of a number of nucleotides at particular positions of the flagellin gene of other Borrelia species. These species-specific peptides are useful as epitopes for the detection of antibodies having specificity for a species-specific flagellin protein, for the detection of T cells or B cells having similar specificity, or as antigens in an immunoassay for the detection of B. lonestari sp. nov. or for the generation of antibodies to be used in an immunoassay.
A fusion protein or peptide comprising a segment of SEQ ID NO:2 having at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids of Table 1 is also an aspect of the present invention. The fusion protein preferably comprises SEQ ID NO:26, however, one skilled in the art, in light of the present disclosure, would be able to construct a number of different fusion proteins from a variety of vectors and the B. lonestari sp. nov. DNA sequences provided herein. It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above. Segments of the flagellin gene may be cloned next to N- and/or C-terminal sequences of genes for other proteins, such as, xcex2-galactosidase or maltose binding protein. A signal peptide that may allow better expression may be optionally included in the fusion protein. It is not necessary that the flagellin protein be transported, however, the signal peptide may help to prevent protease digestion.
A preferred embodiment of the present invention is a method of detecting B. lonestari sp. nov. in a subject. The method comprises the step of contacting a nucleic acid sample from the subject with an oligonucleotide comprising a nucleotide sequence of about 12 to about 30 nucleotides from SEQ ID NO:1 that includes at least one B. lonestari sp. nov.-specific nucleotide or species-specific combination of nucleotides from Table 2 or 3, or a complement thereof, under conditions allowing hybridization to form a duplex, wherein duplex formation indicates the presence of B. lonestari sp. nov. Preferably, the nucleotide sequence comprises the sequence GGTGTTCAAGCG, SEQ ID NO:7 or GTTCAACCAGCT, SEQ ID NO:8. The oligonucleotide may comprise a detectable label and the complex may then be detected by reference to the label.
A further method of detecting B. lonestari sp. nov. in a subject comprises the steps of amplifying a segment of DNA from the subject using a set of PCR(trademark) primers, wherein the segment of DNA includes at least one B. lonestari sp. nov.-specific nucleotide or species-specific combination of nucleotides from Table 2 or 3, and determining the nucleotide sequence of the segment. When the nucleotide sequence of the segment is found in SEQ ID NO:1 or 3, or a complement thereof, then B. lonestari sp. nov. is detected. The PCR(trademark) primers may be designed to be complementary to a region of SEQ ID NO: 1 or 3 or to sequences 5xe2x80x2 and 3xe2x80x2 to any segment to be amplified, and the primers may be complementary to a sequence outside of the herein defined sequences, i.e., in flanking vector or naturally occurring sequences, for example. It is contemplated that regions of as few as 20 or 50 bases may be amplified, or as long as 500 or 1000 bases. One of skill in this art would also understand, in light of the present disclosure, that other means of amplification of DNA or RNA segments would also be applicable to the techniques defined herein.
The present invention also provides a method of detecting B. lonestari sp. nov. in a subject comprising the step of analyzing a DNA sample from the subject for a restriction fragment length polymorphism that is unique to B. lonestari sp. nov. A preferred restriction fragment length polymorphism is from an AluI restriction enzyme digest.
Another embodiment of the present invention is a method of detecting a previously elicited immune response to B. lonestari sp. nov. in a subject. This method may be a cell mediated immunity test. The method comprises the step of contacting a sample from the subject with an epitope having at least a partial amino acid sequence of SEQ ID NO:2 that includes at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids from Table 1, is also an embodiment of the present invention. Contacting of the sample would be under conditions allowing epitope-antibody or epitope-T cell binding to occur to form a complex, and complex formation indicates the presence of a previously elicited immune response to B. lonestari sp. nov. Preferably, the epitope is bound to a detectable label, and a preferred epitope is a flagellin fusion protein. The present inventors also envision the detection of B cells secreting antibody having epitope specificity as defined herein.
A method of detecting B. lonestari sp. nov. in a subject comprising the step of contacting a sample from the subject with an antibody having binding specificity for an epitope having an amino acid sequence from SEQ ID NO:2 that includes at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids from Table 1 is also an embodiment of the present invention. The contacting is under conditions allowing epitope-antibody binding to occur to form a complex and complex formation indicates the presence of B. lonestari sp. nov. Preferably, the epitope has a number of amino acids less than that of SEQ ID NO:2. In these immunoassay procedures, a further step of contacting the complex with a detectably labeled antibody having binding specificity for the complex may be included.
Most preferably, the subject of these detection methods is a human suspected of being infected with B. lonestari sp. nov., although suspected animal reservoirs are also preferred. Any animal that may have been bitten by a tick and that may carry this new spirochete may be tested, including domestic animals such as dogs, cats, cattle, or turkeys, for example.
A test kit for the detection of B. lonestari sp. nov. in a biological sample is also an aspect of the present invention. A kit may comprise in packaged combination; a carrier means adapted to receive a plurality of container means in close confinement therewith; a first container means including an oligonucleotide comprising a nucleotide sequence that includes at least one B. lonestari sp. nov.-specific nucleotide or species-specific combination of nucleotides from Table 2 or 3, or a complement thereof; and at least one microtiter plate. The oligonucleotide may encode all of SEQ ID NO:2 or a portion thereof.
Alternatively, a kit may have a first container means including a first antibody having binding specificity for an epitope, the epitope having a partial or complete amino acid sequence of SEQ ID NO:2 and including at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids from Table 1; and a second container means including a quantity of a detectably labelled antibody having binding specificity for the first antibody.
A further alternative is where a first container means includes a peptide epitope, the epitope being a partial or complete amino acid sequence of SEQ ID NO:2 and including at least one B. lonestari sp. nov.-specific amino acid or species-specific combination of amino acids from Table 1; and a second container means including a quantity of a detectably labelled antibody having binding specificity for immunoglobulin of the biological sample.
In these test kits, the detectably labelled antibody may be an enzyme-linked antibody, a fluorescently tagged antibody, or a radiolabeled antibody. Preferably, the detectably labelled antibody is an enzyme-linked antibody, and the kit further includes a third container means including a quantity of a substrate for the enzyme sufficient to produce a visually detectable product.
A diagnostic kit for determining the presence of B. lonestari sp. nov., in accordance with the present invention, may comprise any one or more of the following components:
1. Unique components in accordance with the present invention:
a. An oligonucleotide complementary to a portion of the flagellin gene or the 16s rRNA gene at a region having a species-specific nucleotide or species-specific combination of nucleotides.
b. Oligonucleotide primers for PCR(trademark) designed to amplify a sequence of SEQ ID NO:1 or 3 where a first primer has a sequence 5xe2x80x2 to a region of SEQ ID NO:1 or 3 having a species-specific nucleotide or species-specific combination of nucleotides and a second primer has a sequence 3xe2x80x2 to the region. Primers may be designed to hybridize outside of the sequences depicted by SEQ ID NO: 1 or 3, since they may be complementary to vector sequences or naturally occurring flanking sequences, for example.
c. A double stranded internal fragment of SEQ ID NO:1 or 3 provided for cloning and DNA sequencing to confirm the identity of a sequenced test fragment.
d. DNA comprising the nucleic acid sequence of SEQ ID NO:1, 3 or 4 as a positive control template DNA for hybridization, sequencing, or RFLP analyses. This DNA may comprise plasmid DNA from clones described in Examples 2 and 3.
e. Antibody having binding specificity for a B. lonestari flagellin species-specific epitope or species-specific combination of epitopes.
f. A peptide having an amino acid sequence that includes a species-specific amino acid or species-specific combination of amino acids of Table 1.
2. Commercially available reagents:
a. Components of a PCRM reaction protocol.
b. Components of a dideoxy-based sequencing protocol.
c. Components of an ELISA protocol.
The following listing provides an identification of those sequences provided with sequence identifiers.
It will be understood that this invention is not limited to the exact nucleic acid and amino acid sequences described herein except for those species-specific nucleotides and amino acids and species-specific combinations of nucleotides and amino acids of Tables 1, 2 and 3. Therefore, DNA segments prepared in accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acid sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.
The process of selecting and preparing a nucleic acid segment which includes a sequence from within SEQ ID NO:1 or 3 may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR(trademark) technology of U.S. Pat. No. 4,603,102 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.